INTRODUCTION TO MOLECULAR BIOLOGY

Musonda Machiko
TMed(KIHERS),Dip. Biomed( EHC), BSc. Biomed(UNZA)
MSc. Biomed(Buenos Aires), MSc. Biomed(Freiburg)
machikoveli@gmail.com
Course outline

Introduction to Molecular Biology

Introduction to the study of gene structure and
function
Recombinant DNA technology
Applied techniques in recombinant DNA technology
The Human Genome Project (HGP)
 Objectives of the course
1. Describe cell function at molecular level.

2. Discuss gene expression regulation in prokaryotes

and eukaryotes.

3. Demonstrate basic gene manipulation and

recombinant DNA techniques and their application in
routine and research work.

4. Apply bioinformatics knowledge to the study of

health and disease conditions
Prescribed textbooks
• Molecular Biology of the Cell (5th edition) (2008) Alberts, B. Bray, D,
Lewis, J, Raff, M, Roberts, K and Watson, J.D. Garland Publishing,
Inc, New York. ISBN-10: 08115341067; ISBN-13 978 0815341062.

• Genomes 3 (2007) Brown, T.A. Garland Science Publishing,

London. ISBN: 0 8153 4138 5.

• Gene Cloning: An Introduction (6th Edition) (2010). Brown, T.A.

Chapman and Hall, London. ISBN-10 1444334077; ISBN-13
1444334074
Molecular Biology is a Synthesis of Several Disciplines
 Definition of Molecular Biology

Molecular Genetics, or Molecular Biology, is the study of the

• biochemical mechanisms of inheritance
• biochemical nature of the genetic material and its control of
phenotype
• connection between genotype and phenotype
Genotype -------> Phenotype
It is the understanding of the molecular basis of biological
processes through studies on the gene
Molecular biology is the branch of biology that studies
the molecular basis of biological activity.
Overview of organizations of the Cell

•Nucleus = library
•Chromosomes = bookshelves
•Genes = books
•Almost every cell in an organism contains the same libraries and
the same sets of books.
•Books represent all the information (DNA) that every cell in the
body needs so it can grow and carry out its various functions
 Overview of organizations of life

• Life is specified by the genome of the myriad of

organisms
• Every organism possesses a genome that contains
biological information needed to construct and maintain a
living example of that organism
• Most genomes are made up of Deoxyribonucleic acid
(DNA) but a few viruses have Ribonucleic acid (RNA)
genomes
• DNA & RNA are polymeric molecules made up of chains
of monomeric subunits called nucleotides
 Some terminologies

•Genome:
An organism’s genetic material.
–Bacteria contains about 600,000 DNA base pairs
–Human and mouse genomes have about 3 billion base pairs
–The human genome has 24 distinct chromosomes.
–Each chromosome contains many genes
•Gene:
A discrete unit of hereditary information located on the chromosomes
and consisting of DNA.
–Basic physical and functional unit of heredity.
–Specific sequences of DNA bases that encode instructions on how to make
proteins
Terminologies cont’d

•Proteins:
–Make up the cellular structure
–large, complex molecules made up of smaller subunits
called amino acids.
•Genotype: The genetic makeup of an organism
•Phenotype: The physical expressed traits of an organism
•Nucleic acid: Biological molecules (RNA or DNA) that
allow organisms to reproduce
All Life depends on 3 critical molecules

•DNA
–Holds information on how cell works
•RNA
–Acts to transfer short pieces of information to different parts of
cell
–Provides templates to synthesize into protein
•Proteins
–Form enzymes that send signals to other cells and regulate
gene activity
–Form body’s major components (e.g. hair, skin, etc.)
 The human genome
The nuclear genome: The mitochondrial genome:

• Comprises approx. 3 200 000 000

nucleotides of DNA
• A circular DNA molecule of 16
• Consists of 24 chromosomes; 22 569 nucleotides
autosomes and two sex
chromosomes (X and Y) • Multiple copies are located in
mitochondria
• Contains 37 genes
• Contains about 35 000 genes
 The human genome cont’d

• Each cell of the adult human body has its own copy of the genome, except for a few
cell types

• Somatic cells – cells that are diploid and contain two copies of each autosome and
two sex chromosomes (46 chromosomes) (most cells of the body)

• Sex cells – cells that are haploid and contain one of each autosome and one sex
chromosome (23 chromosomes)

• Both cell types have about 8 000 copies of the mitochondrial genome
 Genome expression

• The genome is a store of biological information but on its own it is

unable to release that information

• Utilisation of biological information requires genome expression

• Initial product of genome expression is called transcriptome

• Transcriptome – collection of RNA molecules derived from protein-

coding genes with biological information required by the cell at a
particular time
 Genome expression cont’d

• Transcriptome is maintained by the process called

transcription
• The second product of genome expression is the
proteome (cell’s repertoire of proteins)
• The proteins that make up the proteome are
synthesised by translation of individual RNA molecules
present in the transcriptome
 DNA As a Primary Agent of Genetic Material

• In early 1900's many people thought that protein was the genetic material
responsible for inherited characteristics
• One of the reasons behind this belief was the knowledge that proteins
were quite complex molecules and therefore, they must be specified by
molecules of equal or greater complexity (i.e. other proteins)
• DNA was known to be a relatively simple molecule, in comparison to
proteins, and therefore it was hard to understand how a complex
molecule (a protein) could be determined by a simpler molecule (DNA).
• What were the key experiments which identified DNA as the primary
genetic material?
 Transforming Principle

1928 F. Griffith :
• Showed that when Streptococcus pneumonia is injected into mice caused pneumonia
and death in the mouse.
• Occasionally, variants (mutants) of the bacteria arose which had a defect in the
production of the capsular polysaccharide
• The mutants had two characteristics:

The virulent smooth wild type pneumococcus can be heat treated and rendered avirulent
 The experiments:

 Controls:
Wild type (smooth) + mouse = dead mouse
Mutant (rough) + mouse = live mouse
Heat treated wild type (smooth) + mouse = live mouse

 Combinations:

Heat treated wild type (smooth) + mutant (rough) +

mouse = dead mouse
In this case when the bacteria were recovered from the cold lifeless mouse they were smooth
virulent pneumococcus (i.e. indistinguishable from wild type).
A closer look at what is going on, by keeping and keeping track of different subtypes
 Heat treated wild type (smooth) type I + mutant(rough) type II + mouse = dead mouse

In this case when the bacteria were isolated from the cold lifeless mouse they were smooth
virulent type I pneumococcus.
Transforming Principle cont’d
• The overall conclusions from these experiments was that there was a
"transforming agent" in the heat treated type I bacteria which
transformed the live mutant (rough) type II bacteria to be able to
produce type I capsule polysaccharide.

• Question:

Was the "transforming agent" protein or DNA, or what?

To prove this two most important experiments performed by
1. Avery and colleagues
2. Hershey-Chase
1944 Oswald Avery, Colin McLeod & Maclyn McCarty Experiment

• The experiment of Griffith could not be taken further until methods were
developed to separate and purify DNA and protein cellular components
• Avery & Colleagues utilized methods to extract relatively pure DNA from
pneumococcus to determine whether it was the "transforming agent"
observed in Griffith's experiments.

 The experiment:
• Wild type (smooth) type I -> extract the DNA component
• Mutant (rough) type II + type I DNA + mouse = dead mouse
• Isolation of bacteria from the dead mouse showed that they were
type I wild type (smooth) bacteria
A more sophisticated experiment:
• Purified type I DNA was divided into two aliquots.
• One aliquot was treated with DNAse - an enzyme which non-
specifically degrades DNA.
• The other aliquot was treated with Trypsin - a protease which
(relatively) non-specifically degrades proteins.
• Type I DNA + DNAse + mutant (rough) type II + mouse = live mouse
• Type I DNA + Trypsin + mutant (rough) type II + mouse = dead mouse
Conclusion:
• This provided strong evidence that the "transforming agent" was in
fact DNA (and not protein).
• However, not everyone was convinced. Some people felt that a
residual amount of protein might remain in the purified DNA, even
after Trypsin treatment, and could be the "transforming agent”.
1952 Alfred Hershey & Martha Chase
• A. Hershey and M. Chase studied the independent roles of
protein and DNA in bacteriophage T2 infection.

• T2 is a virus that infects bacterial cells:

1.The phage article attaches itself to the bacterial cell wall

2.The phage material is injected into the cell
3.The host cell is induced to produce new phage particles
4.The bacterial cell bursts and hundreds of new phage particles
are released
Hershey & Chase experiment cont’d

• They set up an experiment to monitor the movement of phage

protein and phage DNA during this process

• Labelled each macromolecule separately with radioactive sulfur or

phosphate; protein with 35S and DNA with 32P

• Phages were grown in the presence of 32P and 35S isotopic labels.

• After infection, but prior to cell lysis, the bacteria were whipped up in
a blender and the phage particles were separated from the bacterial
cells. The isolated bacterial cells were cultured further until lysis
occurred. The released progeny phage were isolated
Hershey & Chase experiment cont’d

 Results:
• The majority of 32P entered the cell during infection, whereas the
majority of 35S stayed outside the cell.
• The new phage particles produced by the infected bacterial cell
contained a large percentage of labeled DNA but virtually none of
the labeled protein.
 Conclusion:
• The material which was being transferred from the phage to the
bacteria during infection appeared to be mainly DNA. Although the
results were not entirely unambiguous they provided additional
support for the view that DNA was the "stuff" of genetic inheritance.
References
• Alberts, B, Bray, D, Lewis, J, Raff, M, Roberts, K and
Watson, JD (2008). Molecular Biology of the Cell (5th
edition). Garland Publishing, Inc, New York
• Brown, TA (2007). Genomes 3. Garland Publishing, Inc,
New York
• DNA Interactive: http://www.dnai.org/ Cold Spring Harbor
Laboratory